The objective of this proposal is to elucidate the mechanism(s) underlying regulation of connexin35/36-mediated cell-cell coupling. Connexin35/36 (denoted Cx36 in mammals and Cx35 in other vertebrates) is the most abundant neuronal gap junction protein and is widely expressed in the brain, spinal cord, and retina. The electrical coupling of neurons through gap junctions has been implicated in the etiology of epilepsy (e.g. the gene coding for Cx36 is associated with juvenile myoclonic epilepsy) as well as its pathology. Inhibition of Cx36-mediated coupling can suppress epileptiform activity. Thus understanding the mechanisms that regulate Cx35/36-mediated gap junctional coupling will increase our understanding of the physiological basis of epilepsy, and may identify new targets for future treatments of epilepsy. Very little is known about the regulation of gap junctional coupling in the brain. However, in the retina the coupling state of identified Cx35/36-coupled networks are modulated by endogenous signaling pathways in response to light and neurotransmitter signaling. In cell culture coupling mediated by Cx35 gap junctions is regulated by phosphorylation, and the phosphorylation state of a subpopulation of Cx35 gap junctions in the retina is decreased in light-adapted retinas relative to dark-adapted retinas. This proposal will test the hypothesis that the strength of Cx35-mediated cell-cell coupling in the retina is dynamically modulated by phosphorylation in response to adapting light stimuli and endogenous signaling. Experiments designed to test this hypothesis will be carried out using zebrafish as a model organism. We will use tracer injections to identify a Cx35-coupled cell type and evaluate its degree of coupling under different lighting conditions. Transgenic zebrafish expressing non-phosphorylatable Cx35 will be utilized to test the relationship between phosphorylation of Cx35 and regulated changes in coupling. In addition we will use pharmacological manipulations to dissect which signaling pathways alter Cx35 phosphorylation during light adaptation. PUBLIC HEALTH RELEVANCE: Direct communication between neurons through gap junction proteins is a component of visual processing, is implicated in learning processes in the brain, and is thought to contribute to certain forms of epilepsy. Understanding how this communication is regulated is key to understanding these processes, and may identify new targets for epilepsy treatments.